CRISPR-based methods appear to offer reliable SARS-CoV-2 testing and screening as efficiently as polymerase chain reaction (PCR) techniques, potentially making it possible to capture the leading edge of an outbreak using less expensive technology.
Clustered regularly interspaced short palindromic repeats (CRISPR) is a bacterial immune system component that contains a programmable protein able to cut DNA or RNA. In a study published February 11 in JAMA Network, researchers used a specific CRISPR-based assay, Cas13-based, rugged, equitable, scalable testing (CREST).
CREST was as efficient at detecting SARS-CoV-2 infections in asymptomatic participants as the U.S. Centers for Disease Control and Prevention (CDC)-recommended reverse transcription–quantitative polymerase chain reaction (RT-qPCR) technology, considered the gold-standard testing method, the researchers indicated.
This is important because PCR SARS detection has faced some challenges. The high demand for COVID-19 testing has overwhelmed supply chains, limiting the availability of critical reagents and specialized equipment necessary for RT-qPCR, the investigators wrote.
On the other hand, CRISPR-based assays offer a robust and sensitive alternative for detecting SARS-CoV-2 genomes. These assays use common and widely available reagents and are adaptable to minimal instrumentation and infrastructure.
In addition to its efficiency, CRISPR makes possible an easy-to-interpret and dependable binary readout, indicating the emission of fluorescence or no fluorescence. The CRISPR-based assay was in perfect concordance with positive cases diagnosed in a CLIA-certified laboratory -- Pacific Diagnostics Laboratory -- further corroborating its robustness, according to Jennifer Rauch, PhD, of the department of molecular, cellular, and developmental biology at the University of California, Santa Barbara (UCSB) and lead author of the study.
Specifically, CREST was designed to be a low-cost and accessible method, offering an option for communities where resources are limited and where access to testing is difficult, the scientists said. Moreover, CRISPR technology is scalable, enabling high-throughput testing. Also, it uses laboratory-made or off-the-shelf commercially available reagents, minimizing supply chain issues.
The investigators surmised that CREST can offer a solution for COVID-19 screening locations where access to professional laboratories is restrictive, and where a high volume of repetitive sampling is necessary, such as at universities.
Their research was based on how reopening colleges and universities during the COVID-19 pandemic is a significant public health challenge, and that utilizing accessible practical techniques for detecting SARS-CoV-2 among students would be a key component of recurrent surveillance testing for detecting, containing, and alleviating the virus.
The UCSB investigators and colleagues elsewhere undertook an observational study that assessed CRISPR-based techniques for screening and detecting SARS-CoV-2 among asymptomatic college students.
From UCSB, 1,808 healthy asymptomatic college students were screened for SARS-CoV-2 using RT-qPCR and CRISPR-based assays. The results were obtained from May 28 to June 11, 2020, approximately two months into a statewide stay-at-home mandate, and from June 23 to July 2, 2020, approximately three weeks after local restrictions for isolation were eased.
RT-qPCR detected nine samples positive for SARS-CoV-2, while eight were confirmed using CRISPR-CREST and clinical laboratory diagnostic testing, uncovering a change in viral prevalence that coincided with the relaxation of lockdown measures and the rise of COVID-19 cases in the community.
While there weren't any COVID-19 cases in the study population during the May to June collection period, the investigators found a substantial shift in prevalence approximately one month later, which coincided with changes in community restrictions and public interactions.
According to the researchers, the CRISPR-based assay performed as well as the CDC-recommended RT-qPCR assay.
"Our study substantiates the utility of self-collected oropharyngeal swabs and CRISPR-based testing as valuable alternatives for large-scale surveillance sampling of SARS-CoV-2 in asymptomatic individuals," they wrote.
Both methods detected two sites in the nucleocapsid gene, N1 and N2, and one site in the host RNaseP transcript, which ensured consistency in the analyses, the researchers stated.
The scientists indicated that they performed RT-qPCR by following the procedures in the emergency use authorization (EUA) granted by the U.S. Food and Drug Administration (FDA). CREST has not yet received an EUA from the FDA.